HO-1在小鼠内毒素急性肺损伤中的作用:与调控线粒体质量控制的关系  

Role of HO-1 in endotoxin-induced acute lung injury in mice:relationship with regulation of mitochondrial quality control

作　　者：史佳[1] 刘华洋 刘沙沙[1] 张蓝天 武雅 余剑波[1] Shi Jia;Liu Huayang;Liu Shasha;Zhang Lantian;Wu Ya;Yu Jianbo(Department of Anesthesiology and Critical Care Medicine,Nankai Clinical College of Tianjin Medical University(Tianjin Nankai Hospital),Tianjin 300100,China)

机构地区：[1]天津医科大学南开临床学院(天津市南开医院)麻醉科与重症医学科,天津300100

出　　处：《中华麻醉学杂志》2022年第5期600-605,共6页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金青年项目(82004076);天津市卫生健康科技项目青年项目(QN20027)。

摘　　要：目的评价血红素氧合酶-1(HO-1)在小鼠内毒素急性肺损伤中的作用及其与调控线粒体质量控制的关系。方法清洁级健康雄性成年C57BL/6小鼠,基于CRISPER/Cas9开发的EGE系统制备HO-1诱导性基因敲除小鼠,并构建HO-1过表达腺病毒载体诱导HO-1基因过表达小鼠,6~8周龄,体重20~25 g,采用随机数字表法,将野生型C57BL/6(WT)、HO-1基因敲除型小鼠(HO-1^(-/-))和HO-1基因过表达型小鼠(HO-1^(+/+))分别分为2组(n=6):对照组(WT组、HO-1^(-/-)组和HO-1^(+/+)组)和内毒素急性肺损伤组(ALI组、HO-1^(-/-)+ALI组和HO-1^(+/+)+ALI组)。经尾静脉注射LPS 15 mg/kg制备小鼠内毒素急性肺损伤模型,各对照组给予等容量生理盐水。给予LPS或生理盐水后12 h时处死小鼠取肺组织,光镜下观察肺组织病理学结果并进行肺损伤评分,测定肺组织还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量,计算GSH/GSSG比值,透射电镜下观察线粒体超微结构,测定线粒体膜电位(MMP)水平,采用Western blot法测定HO-1、线粒体质量控制相关蛋白线粒体融合蛋白2(Mfn2)、动力相关蛋白1(Drp1)、过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)、核呼吸因子1(NRF1)、线粒体自噬标志蛋白PTEN诱导激酶1(PINK1)和E3泛素-蛋白连接酶(Parkin)的表达,观察小鼠12 h生存情况。结果与对照组(WT组、HO-1^(-/-)组和HO-1^(+/+)组)比较,内毒素急性肺损伤组(ALI组、HO-1^(-/-)+ALI组和HO-1^(+/+)+ALI组)小鼠12 h生存率和MMP降低,肺损伤评分增加,GSH含量和GSH/GSSG比值降低,GSSG含量升高(P<0.05);与ALI组比较,HO-1^(-/-)+ALI组小鼠12 h生存率和MMP降低,肺损伤评分增加,GSH含量和GSH/GSSG比值降低,GSSG含量升高,HO-1、Mfn2、PGC-1α、NRF1、PINK1和Parkin表达下调,Drp1表达上调,HO-1^(+/+)+ALI组小鼠12 h生存率和MMP升高,肺损伤评分降低,GSH含量和GSH/GSSG比值升高,GSSG含量降低,HO-1、Mfn2、PGC-1α、NRF1、PINK1和Parkin表达上调,Drp1表达下�Objective To evaluate the role of heme oxygenase-1(HO-1)in endotoxin-induced acute lung injury(ALI)and the relationship with the regulation of mitochondrial quality control in mice.Methods Clean-grade healthy male adult C57BL/6 mice,aged 6-8 weeks,weighing 20-25 g,were selected.HO-1 inducible gene knockout mice(HO-1^(-/-))were prepared based on CRISPER/Cas9-mediated EGE system,and HO-1 gene overexpression mice(HO-1^(+/+))were prepared by transfection of HO-1 overexpressed adenovirus vector.The mice were divided into 2 groups(n=6 each)using a random number table method:control group(group WT,group HO-1^(-/-),group HO-1^(+/+))and endotoxin-induced ALI group(group ALI,group HO-1^(-/-)+ALI,group HO-1^(+/+)+ALI).Lipopolysaccharide 15 mg/kg was injected through the tail vein to develop the model of endotoxin-induced ALI,and the equal volume of normal saline was given instead in each control group.The mice were sacrificed by bloodletting at 12 h after lipopolysaccharide or normal saline administration.The lung tissues were harvested for microscopic examination of the pathological changes which were scored,for determination of GSH and GSSG contents,for observation of the ultrastructure of mitochondria(with a transmission electron microscope)and survival within 12 h,for measurement of mitochondrial membrane potential(MMP)levels,and for determination of the expression of mitochondrial quality control-related proteins mitochondrial fusion protein 2(Mfn2)and dynamin-related protein 1(Drp1),peroxisome proliferator-activated receptor gamma coactivator 1α(PGC-1α),nuclear respiratory factor 1(NRF1),mitophagy marker protein PTEN-induced kinase 1(PINK1)and E3 ubiquitin-protein ligase Parkin.The ratio of GSH/GSSG was calculated.Results Compared with control group(group WT,group HO-1^(+/+)and group HO-1^(-/-)),the 12-h survival rate and MMP were significantly decreased,the lung injury score was increased,GSH content and GSH/GSSG ratio were decreased,and the content of GSSG was increased in endotoxin-induced ALI groups(group ALI,gr

关 键 词：内毒素血症 急性肺损伤 血红素氧合酶-1 线粒体质量控制 

分 类 号：R563[医药卫生—呼吸系统]